Zinc is essential for all organisms. Zinc is a catalytic cofactor of over 300 enzymes and is a critical component of structural motifs such as zinc fingers. Therefore, cells require efficient mechanisms for the accumulation of zinc. Many zinc-dependent proteins are either secreted or localized to the interior of intracellular organelles such as those of the secretory pathway and mitochondria. Therefore, efficient mechanisms are also needed to distribute zinc into intracellular compartments. In contrast, excess zinc can be toxic to cells. This toxicity may be due to the binding of zinc to inappropriate sites in proteins or cofactors. For example, excess zinc can interfere with mitochondrial aconitase activity and impair respiration. The essential yet toxic nature of zinc necessitates precise homeostatic mechanisms to control the intracellular levels of "free", i.e., loosely bound or labile, zinc. We currently know little about intracellular zinc transport and zinc sequestration. We are studying these processes using the yeast Saccharomyces cerevisiae as a model eukaryotic cell. Our studies have raised four central hypotheses that are the foundation of this proposal: 1) The yeast vacuole, the lysosome-like compartment of the yeast cell, plays an important role in storing zinc in replete cells for later use under conditions of zinc deficiency. 2) The vacuole acts as a zinc buffer to control zinc levels in the cytosol and other compartments when the intracellular labile zinc pool changes in response to perturbations in zinc homeostasis. 3) The Msc2 protein, a member of the ubiquitous CDF family of metal ion transporters, is responsible for transporting zinc into the endoplasmic reticulum to supply the metal to secreted and resident proteins in the secretory pathway. 4) Additional transporters are also present in the ER of yeast that transport zinc into the secretory pathway. To test these hypotheses, we propose a multifaceted approach, using a synergistic combination of genetic, molecular biology, cell biology, biochemistry, and bioinorganic chemistry tools, to define the mechanisms and roles of intracellular zinc transport in yeast. [unreadable] [unreadable]